The objective of the current project is to obtain a clear understanding of the mechanisms involved in the maternal-to-fetal transfer of folate, an essential nutrient for normal growth and development of the feto- placental unit. The essential role of folate in fetal development is underscored by recent findings that supplementation of the vitamin during the periconceptional period decreases the incidence of neural tube defects in developing fetus. The nutritional needs of the fetus for folate have to be met solely by transplacental transfer from the mother and therefore a thorough understanding of the molecular mechanisms involved in this process is warranted. Transport of folate across the placenta occurs in two steps - entry of folate from the maternal circulation into the syncytiotrophoblast across the maternal-facing brush border membrane and exit of folate from the syncytiotrophoblast into the fetal compartment across the fetal-facing basal membrane. It is proposed that the first step, i.e. entry into the syncytiotrophoblast, utilizes a novel cellular pathway called potocytosis. According to the proposed model, this pathway involves unique membrane structures known as caveolae and enables an efficient coupling among the three molecular components participating in folate entry, namely V-type H+ -pump, folate receptor, and folate transporter. The function of the folate receptor is to capture folate from the maternal circulation and concentrate it in caveolae. The folate transporter transports folate from the caveolar space into the cytoplasm, and this process is energized by a transmembrane H+ gradient. The role of the H+ - pump is to generate this driving force. Experimental support for the model will be obtained by characterizing the molecular and functional nature of these components and by demonstrating colocalization of the components with caveolin, the caveola-specific protein, in the brush border membrane of the syncytiotrophoblast. The folate receptor and the folate transporter have been cloned from human placenta and the availability of the cDNAs facilitates the proposed studies. Functional characterization of these cloned proteins will be carried out by expressing the respective cDNAs in mammalian cells which possess no or little endogenous folate receptor or folate transporter. The gene for the folate transporter has been localized to human chromosome 21q22.3, a region believed to be critical in the pathogenesis of the clinical features of Down Syndrome (Trisomy 21). Studies are proposed to elucidate the structure and organization of the folate transporter gene. The mechanisms by which folate crosses the basal membrane are not known. It is likely that the folate transporter is also expressed in this membrane, facilitating the exit process. Proposed studies will characterize the transport mechanism available for folate in isolated basal membrane vesicles and also determine the metabolic fate of folate within the syncytiotrophoblast. Cultured trophoblast cells and choriocarcinoma cells will be used as model systems to investigate the regulation of folate transport in placenta by hormones, folate availability and alcohol.